Phosphinic acid derivatives with metallopeptidase inhibitory activity

ABSTRACT

Compounds of formula (I), ##STR1## wherein R is a biphenyl group optionally substituted, by one or more substituents, the same or different selected among halogen atoms, hydroxy groups, alkoxy, alkyl thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C 1  -C 3  alkyl groups containing one or more fluorine atoms, carboxy groups, nitro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, momo- or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylamino-carbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety; R 1  is a hydrogen atom, a straight or branched C 1  -C 6  alkyl group or an arylalkyl group having from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is a phenyl, a biphenyl, a napthyl or a 5 or 6 membered aromatic heterocycle with one or two heteroatoms selected among nitrogen, oxygen and sulphur, optionally substituted with one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C 1  -C 3  alkyl groups containing one or more fluorine atoms, carboxy groups, nitro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety; R 2  is a straight or branched C 1  -C 6  alkyl group, optionally containing one or more fluorine atoms or one or more --NH-- groups, an arylalkyl, an arylcarbonylaminoalkyl, an arylalkylcarbonylaminoalkyl or an arylaminocarbonylalkyl group having from 1 to 6 carbon atoms and optionally one or more --NH-- groups in the alkyl moiety, the aryl being optionally substituted by one or more substituents, the same or different, selected among halogen atoms, hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, C 1  -C 3  alkyl groups containing one or more fluorine atoms, carboxy groups, nitro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono-or di-alkylamino groups having from 1 to 6 carbon atoms in the alkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety; m is 0 or 1; X is a hydrogen or fluorine atom; the carbon atom marked with an asterisk is an asymmetric carbon atom; and their pharmaceutically acceptable salts, are described. The compounds of formula (I) are endowed with a mixed ACE-inhibitory and NEP-inhibitory activity and are useful in the treatment of cardiovascular diseases.

The present invention relates to phosphinic acid derivatives and, moreparticularly, it relates to phosphinic acid derivatives useful in thetreatment of cardiovascular diseases as metallopeptidase inhibitors.

The pharmacologic interest towards the study of metallopeptidaseinhibitory molecules derives from the role that said enzymes exert onthe level of the cardiocirculatory system.

It is well-known, in fact, that compounds with angiotensin convertingenzyme (ACE) inhibitory activity are mainly useful in the treatment ofhypertension and of heart failure in that they inhibit the formation ofangiotensin II, a substance which increases the blood pressure.

Compounds with endothelin converting enzyme (ECE) inhibitory activityare useful as anti-vasoconstrictors in that they inhibit the formationof endothelin, a 21 amino acid peptide with vasoconstrictor activity.

Instead, compounds with inhibitory activity of the neutral endopeptidaseenzyme (NEP), also called enkephalinase, are useful as vasodilators inthat the NEP enzyme is responsible for the inactivation, not only ofendogenous enkephaline, but also of atrial natriuretic factor (ANF), avasodilator hormone secreted by heart.

Therefore, even exerting their action on the cardiovascular system withdifferent mechanisms of action, the compounds with metallopeptidaseinhibitory activity are generally used, alone or in combination, in thetreatment of hypertension, renal failure and congestive heart failure.

In the U.S. Pat. No. 4,396,772 (Squibb & Sons, Inc.) some phosphinicacid derivatives and, more particularly, some phosphinyl alkanoyl aminoacids are described as ACE-inhibitors.

As described by Brandley P. Morgan et al. in Journal of the AmericanChemical Society, 1991, 113, 297-307, some phosphinic acid derivativesand, particularly, the compound of formula ##STR2## wherein Ph is aphenyl group, resulted to be endowed with inhibitory activity towardsthermolysin, an endopeptidase of bacterial origin. Successively, asdescribed by Stephen R. Bertenshaw et al. in Journal of MedicinalChemistry, 1993, 36, 173-176, some phosphinic acid derivatives having adipeptide moiety analogous to that of phosphoramidon, a well-knownmolecule with ECE-inhibitory activity, resulted to be devoid of anysignificant ECE-inhibitory activity. Now we have found phosphinic acidderivatives which are endowed with angiotensin converting enzyme as wellas neutral endopeptidase enzyme inhibitory activity (mixed ACE/NEPinhibitory activity) which renders them particularly useful in thecardiovascular therapy. Therefore, object of the present invention arethe compounds of formula ##STR3## wherein

R is a biphenyl group optionally substituted by one or moresubstituents, the same or different, selected among halogen atoms,hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups havingfrom 1 to 6 carbon atoms in the alkyl moiety, C₁ -C₃ alkyl groupscontaining one or more fluorine atoms, carboxy groups, nitro groups,amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups,mono- or di-alkylamino groups having from 1 to 6 carbon atoms in thealkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6carbon atoms in the alkyl moiety;

R₁ is a hydrogen atom, a straight or branched C₁ -C₆ alkyl group or anarylalkyl group having from 1 to 6 carbon atoms in the alkyl moietywherein the aryl is a phenyl, a biphenyl, a naphthyl or a 5 or 6membered aromatic heterocycle with one or two heteroatoms selected amongnitrogen, oxygen and sulphur, optionally substituted with one or moresubstituents, the same or different, selected among halogen atoms,hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups havingfrom 1 to 6 carbon atoms in the alkyl moiety, C₁ -C₃ alkyl groupscontaining one or more fluorine atoms, carboxy groups, nitro groups,amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups,mono- or di-alkylamino groups having from 1 to 6 carbon atoms in thealkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6carbon atoms in the alkyl moiety;

R₂ is a straight or branched C₁ -C₆ alkyl group, optionally containingone or more fluorine atoms or optionally one or more --NH-- groups, anarylalkyl, an arylcarbonylaminoalkyl, an arylalkylcarbonylaminoalkyl oran arylaminocarbonylalkyl group having from 1 to 6 carbon atoms andoptionally one or more --NH-- groups in the alkyl moiety, the aryl beingoptionally substituted by one or more substituents, the same ordifferent, selected among halogen atoms, hydroxy groups, alkoxy, alkyl,thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms inthe alkyl moiety, C₁ -C₃ alkyl groups containing one or more fluorineatoms, carboxy groups, nitro groups, amino or aminocarbonyl groups,acylamino groups, aminosulphonyl groups, mono- or di-alkylamino groupshaving from 1 to 6 carbon atoms in the alkyl moiety, mono- ordi-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in thealkyl moiety;

m is 0 or 1;

X is a hydrogen or fluorine atom;

the carbon atom marked with an asterisk is an asymmetric carbon atom;

and their pharmaceutically acceptable salts.

Object of the present invention are the compounds of formula I in theform of stereoisomeric mixture as well as in the form of singlestereoisomers.

The compounds of formula I object of the present invention are endowedwith a mixed ACE-inhibitory and NEP-inhibitory activity and are usefulin the treatment of cardiovascular diseases.

In the present description, unless otherwise specified, with the termbiphenyl group we intend a 2-biphenyl, 3-biphenyl or 4-biphenyl group;with the term alkyl group we intend a straight or branched alkyl such asmethyl, ethyl, n.propyl, isopropyl, n.butyl, sec-butyl, tert-butyl,isobutyl, n.pentyl, 2-pentyl, 3-pentyl, isopentyl, tert-pentyl, n.hexyland isohexyl; with the term halogen atom we intend a fluorine, chlorine,bromine or iodine atom; with the term acyl we intend an acyl groupderiving from an aliphatic or aromatic carboxylic acid such as acetic,propionic, butyric and benzoic acid; with the term aryl group we intendan aromatic group such as phenyl, 2-biphenyl, 3-biphenyl, 4-biphenyl,1-naphthyl and 2-naphthyl or a 5 or 6 membered heterocyclic groupcontaining 1 or 2 heteroatoms selected among nitrogen, oxygen andsulphur such as thiazole, isoxazole, oxazole, isothiazole, pyrazole,imidazole, thiophene, pyrrole, pyridine, pyrimidine and furan,optionally benzocondensed. Examples of pharmaceutically acceptable saltsof the compounds of formula I are the salts with alkali or alkali-earthmetals and the salts with pharmaceutically acceptable organic bases.

Preferred compounds of formula I are the compounds wherein R is abiphenyl group optionally substituted with from 1 to 3 substituents, thesame or different, selected among chlorine or fluorine atoms or hydroxygroups; R₁ is a straight or branched C₁ -C₆ alkyl group or an arylalkylgroup having from 1 to 6 carbon atoms in the alkyl moiety wherein thearyl group is a phenyl or a biphenyl; R₂ is a straight or branched C₁-C₆ alkyl, an arylcarbonylaminoalkyl, an arylalkylcarbonylaminoalkyl oran arylalkyl group having from 1 to 6 carbon atoms in the alkyl moietywherein the aryl group is a phenyl, m is 1 and X is hydrogen.

Still mope preferred compounds of formula I are the Compounds wherein Ris a 4-biphenyl group; R₁ is a straight or branched C₃ -C₆ alkyl group;R₂ is an arylcarbonylaminoalkyl or an arylalkylcarbonylaminoalkyl grouphaving from 1 to 3 carbon atoms in the alkyl moiety wherein the arylgroup is a phenyl; m=1 and X=H.

Preferred examples of pharmaceutically acceptable salts of the compoundsof formula I are the salts with alkali metals such as sodium, lithiumand potassium.

The preparation of the compounds of formula I, object of the presentinvention, comprises the reaction between a compound of formula ##STR4##wherein

R₁, R₂, m and X have the above reported meanings and Y represents aprotective group, preferably a C₁ -C₄ alkyl, a phenyl or a phenylalkylgroup having from 1 to 4 carbon atoms in the alkyl moiety; and abiphenylalanine derivative of formula ##STR5## wherein

R has the above reported meanings.

The condensation is carried out according to conventional techniques ofthe chemistry of peptides.

Before carrying out the reaction, it can be useful to properly protectthe optional functional groups which could interfere in the reaction.

The optional protection is carried out according to conventionaltechniques.

For instance, it can be useful to protect the free carboxy function ofthe compound of formula III analogously to the OH function of thephosphinic group.

The evaluation of the usefulness of the optional protection as well asthe selection of the kind of adopted protection, according to thereaction to be carried out and to the functional groups to be protected,are within the normal knowledge of the man skilled in the art.

The removal of the optional protective groups is carried out accordingto conventional techniques.

For a general reference to the use of protective groups in organicchemistry see Theodora W. Greene and Peter G. M. Wuts "Protective Groupsin Organic Synthesis", John Wiley & Sons, Inc.

The compounds of formula II and III are known or easily preparedcompounds according to known methods.

For instance, the compounds of formula II wherein m=1 and X=H can beprepared by reacting a phosphorylated derivative of formula ##STR6##wherein

R₂ and Y have the above reported meanings; with an acrylic acidderivative of formula ##STR7## wherein

R₁ has the above reported meanings and R₃ represents a hydrogen atom or,preferably, a protective group selected among a C₁ -C₄ alkyl, a phenylor a phenylalkyl having from 1 to 4 carbon atoms in the alkyl moiety.

The compounds of formula II wherein m=1 and X=F can be prepared byreacting a compound of formula ##STR8## wherein

R₂ and Y have the above reported meanings; with a α-halo-acid derivativeof formula ##STR9## wherein

R₁ has the above reported meanings, Z is a chlorine or bromine atom andR₃ represents a hydrogen atom or, preferably, a protective groupselected among a C₁ -C₄ alkyl, a phenyl or a phenylalkyl group havingfrom 1 to 4 carbon atoms in the alkyl moiety; in a basic medium.

The compounds of formula VI, in their turn, can be prepared from thecorresponding phosphorylated derivatives of formula IV by treatment withsodium hydride and chlorodifluoromethane (CHF₂ Cl).

The compounds of formula II wherein m=0 can be prepared as described inTetrahedron Letters, 1984, 25, 4737-4740.

Alternatively, the compounds of formula I wherein m=1 and X=H, object ofthe present invention, can be prepared by reacting a phosphorylatedderivative of formula IV with a compound of formula ##STR10## wherein

R and R₁ have the above reported meanings.

The compounds of formula VIII, in their turn, can be prepared byreacting an acrylic acid derivative of formula V wherein R₃ represents ahydrogen atom with a biphenylalanine derivative of formula III.

As previously pointed out, before carrying out said reactions it can beuseful to properly protect the optional functional groups which couldinterfere in the reactions; the removal of the optional protectivegroups is carried out according to conventional techniques. Thecompounds of formula I in the form of single stereoisomers are preparedby stereoselective synthesis or by separation of the stereoisomericmixture according to conventional techniques.

Also the preparation of the salts of the compounds of formula I, objectof the invention, is carried out according to conventional techniques.

The compounds of formula I object of the present invention are endowedwith a mixed ACE-inhibitory and NEP-inhibitory activity and are usefulin the treatment of cardiovascular diseases.

The inhibitory activity of the compounds of formula I was evaluated bymeans of in vitro tests (example 24).

The inhibitory activity of the compounds of formula I, expressed as IC₅₀value, is pharmacologically significant in that it results at nMconcentrations.

For a practical use in therapy, the compounds of formula I can beformulated in solid or liquid pharmaceutical compositions, suitable toopal or parenteral administration.

The pharmaceutical compositions containing a therapeutically effectiveamount of a compound of formula I in admixture with a carrier forpharmaceutical use are, therefore, a further object of the presentinvention.

Specific examples of pharmaceutical compositions according to thepresent invention are tablets, coated tablets, capsules, granulates,solutions and suspensions suitable to opal administration, solutions andsuspensions suitable to parenteral administration.

The pharmaceutical compositions object of the present invention areprepared according to conventional techniques.

For a practical use in therapy it can be useful to formulate thecompounds of formula I in the form of pro-drugs, namely, of derivativeswhich enable the in vivo release of the pharmacologically activemolecule that is to say of the corresponding compounds of formula I.

Specific examples of pro-drugs of the compounds of formula I are theesters of the carboxy group or of the phosphinic group.

The pro-drugs of the compounds of formula I, therefore, are within thescope of the present invention.

The daily dose of the compound of formula I or of the correspondingpro-drug will depend on different factors such as the seriousness of thedisease, the individual response of the patient or the kind offormulation but it is usually comprised between 0.1 mg and 50 mg per Kgof body weight divided into a single dose or into more daily doses.

With the aim of better illustrating the present invention the followingexamples are now given.

EXAMPLE 1

Preparation of ethyl 2-ethoxycarbonyl-4-methylvalerate

Diethyl malonate (20 g; 124.8 mmoles) was added to a mixture of metallicsodium (2.87 g; 124.8 mmoles) dissolved in absolute ethanol (60 ml) at50° C.

After 1 hour at room temperature isobutyl bromide 97% (16.78 g; 118.8mmoles) was added dropwise.

After 2.5 hours at reflux temperature, the reaction mixture was kept atroom temperature overnight.

After adding hydrochloric acid 1N up to pH 4-5, ethanol was evaporatedand the residue was extracted with ethyl ether. After drying andevaporating the solvent at reduced pressure, the crude oil was purifiedby distillation at 78° C./2.5 mmHg after the removal of the exceedingdiethyl malonate at 65° C./7 mmHg. Ethyl2-ethoxycarbonyl-4-methylvalerate (14.4 g; 56% yield) was obtained as acolorless oil.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.87 (d, 6H); 1.21 (t, 6H); 1.52 (m,1H); 1.75 (t, 2H); 3.47 (t, 1H); 4.15 (q, 4H).

EXAMPLE 2

Preparation of 2-ethoxycarbonyl-4-methylvaleric acid

A mixture of potassium hydroxide at 85% (3.6 g; 57.34 moles), water (10ml) and ethanol (10 ml) was heated at 70° C. and hence ethyl2-ethoxycarbonyl-4-methylvalerate (11.75 g; 54.34 mmoles), prepared asdescribed in example 1, was therein added.

After keeping the reaction mixture at reflux temperature for 2 hours,ethanol was evaporated and the resultant solution was washed with ethylacetate and acidified with concentrated hydrochloric acid.

After extracting with ethyl acetate, drying and evaporating the solventat reduced pressure, 2-ethoxycarbonyl-4-methylvaleric acid (6.7 g; 65.5%yield) was obtained as a colorless oil.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.90 (d, 6H); 1.25 (t, 3H); 1.60 (m,1H); 1.79 (t, 2H); 3.44 (t, 1H); 4.19 (g, 2H).

EXAMPLE 3

Preparation of ethyl 2-isobutylacrylate

A mixture of 2-ethoxycarbonyl-4-methylvaleric acid (6.7 g; 36 mmoles),prepared as described in example 2, piperidine (36 mmoles) andparaformaldehyde (1.08 g; 36 mmoles) in pyridine (6.48 ml) was heatedunder reflux for three hours.

After adding water (50 ml) and keeping under reflux for further twohours, the solution was diluted with water and extracted three timeswith ethyl ether.

The organic phase was washed twice with acidic water, dried andevaporated to dryness.

Ethyl 2-isobutylacrylate (3.2 g; 57% yield) was obtained as an oil.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.85 (d, 6H); 1.26 (t, 3H); 1.75 (m,1H); 2.15 (d, 2H); 4.16 (q, 2H); 5.44 (d, 1H); 6.12 (d, 1H).

EXAMPLE 4

Preparation of methyl 4-phenylbutylphosphinic acid

A mixture of 4-phenylbutylphosphinic acid (10 g; 50.4 mmoles), preparedas described in Tetrahedron Letters, 27, 1751-1754, 1986, and trimethylphosphite (83 ml; 706 moles) was heated at 60° C. for 8 hours.

By distilling the reaction mixture at reduced pressure (0.2 mmHg, b.p.125°-127° C.), methyl 4-phenylbutylphosphinic acid (8.9 g; 83% yield)was obtained as a colorless oil.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 1.50-1.90 (m, 6H); 2.61 (t, 2H); 3.74(d, 3H); 5.69, 8.32 (dt, 1H); 7.10-7.33 (m, 5H).

EXAMPLE 5

Preparation of benzyl (4-phenylbutyl)(hydroxy)phosphinyl!acetate

Trimethylsilyl chloride (1.39 ml; 11 mmoles) and benzyl bromoacetate(0.86 ml) were added, at 0° C. and under nitrogen, to a solution of4-phenylbutylphosphinic acid (1 g; 5 mmoles) and triethylamine (11mmoles) in chloroform (20 ml).

The reaction mixture was kept under stirring for 16 hours at roomtemperature and was poured in water (10 ml) and concentratedhydrochloric acid (3.5 ml).

The mixture was extracted with methylene chloride and the organic phasewas washed with brine and dried over sodium sulphate.

The solvent was evaporated at reduced pressure and the resultant oil wastreated with a hexane: ethyl ether=1:1 mixture and filtered affordingbenzyl (4-phenylbutyl)(hydroxy)phosphinyl!acetate (1.05 g; 61% yield) asa white solid.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 1.50-2.00 (m, 6H); 2.55 (bt, 2H);2.95 (d, 2H); 5.12 (s, 2H); 7.06-7.38 (m, 10H).

EXAMPLE 6

Preparation of (4-phenylbutyl)(methoxy)phosphinyl!acetic acid

A solution of benzyl (4-phenylbutyl)(hydroxy)phosphinyl!acetate (1.05 g;3 moles), prepared as described in example 5, in methylene chloride (10ml) was treated with a slight excess of an etheral solution ofdiazomethane up to the formation of a persistent yellow solution.

The solution was treated with acetic acid up to decoloration and thesolvent was evaporated under vacuum.

The resultant etude oil (1.16 g) was dissolved in methanol (100 ml) andhydrogenated into a Parr apparatus in the presence of palladium oncharcoal at 10% (0.3 g).

By filtering off the catalyst and evaporating the reaction mixture undervacuum, (4-phenylbutyl)(methoxy)phosphinyl!acetic acid (0.725 g; 87%yield) was obtained as a colorless oil.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 1.50-2.10 (m, 6H); 2.60 (bt, 2H);2.92 (d, 2H); 3.74 (d, 3H); 7.08-7.31 (m, 5H).

EXAMPLE 7

Preparation of methyl 3-(3-phenylpropyl)(methoxy)phosphinyl!-2-isobutyl-propionate

A solution of sodium (0.345 g; 15 mmoles) in methanol (4 ml) was addeddropwise in three hours, at 0° C. and under nitrogen, to a solution ofmethyl 3-phenylpropylphosphinic acid (2.97 g; 15 mmoles), prepared asdescribed in J. Med. Chem. 1989, 32, 1652-1661, and ethyl2-isobutylacrylate (2.65 g; 17 mmoles), prepared as described in example3.

After one night at room temperature, the reaction mixture was dilutedwith water, acidified with diluted hydrochloric acid up to neutral pHand extracted twice with ethyl acetate.

The organic phase was washed twice with an aqueous solution of potassiumbicarbonate, dried and evaporated to dryness.

The crude residue (2.7 g) was purified by silica gel columnchromatography (eluent CH₂ Cl₂ :CH₃ OH═95:5) affording methyl 3-(3-phenylpropyl)(methoxy)phosphinyl!-2-isobutyl-propionate (2 g; 40.6%yield).

¹ H-NMR (300 MHz, CDCl₃): δ (ppm): 0.87 (d, 3H); 0.90 (d, 3H); 1.30 (m,1H); 1.42-1.95 (m, 6H); 2.14 (m, 1H); 2.68 (t, 2H); 2.77 (m, 1H);3.60-3.71 (2d, 3H); 3.65 (s, 3H); 7.10-7.35 (m, 5H).

By working in a similar way and by using methyl 4-phenylbutylphosphinicacid as starting material, prepared as described in example 4, thefollowing compound was prepared:

methyl 3- (4-phenylbutyl)(methoxy)phosphinyl!-2-isobutyl-propionate

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.88 (dd, 6H); 1.15-1.81 (m, 10H);2.00-2.25 (m, 1H); 2.61 (t, 2H); 2.75 (m, 1H); 3.63 (dd, 3H); 3.67 (s,3H); 7.07-7.31 (m, 5H).

EXAMPLE 8

Preparation of 3-(3-phenylpropyl)(methoxy)phosphinyl!-2-isobutyl-propionic acid

A solution of methyl 3-(3-phenylpropyl)(methoxy)phosphinyl!-2-isobutyl-propionate (0.778 g;2.29 mmoles), prepared as described in example 7, and sodium hydroxide1N (2.29 ml; 2.29 mmoles) in methanol (15 ml), under nitrogen, was keptat 60° C. for 2 days.

After diluting with water and washing with ethyl acetate, the aqueousphase was acidified and extracted with ethyl acetate.

The organic phase, after drying and evaporating the solvent to dryness,furnished 3- (3-phenylpropyl)(methoxy)phosphinyl!-2-isobutyl-propionicacid (0.6 g; 80.4% yield).

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.89 (2d, 6H); 1.28 (m, 1H);1.50-1.20 (m, 7H); 2.20 (m, 1H); 2.67 (t, 1H); 2.75 (m, 1H); 3.68 (2d,3H); 7.10-7.35 (m, 5H).

By working in a similar way the following compound was prepared:

3- (4-phenylbutyl)(methoxy)phosphinyl!-2-isobutyl-propionic acid

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.90 (m, 6H); 1.10-1.90 (m, 10H);2.05-2.35 (m, 1H); 2.60 (t, 2H); 2.78 (m, 1H); 3.68 (d, 3H); 7.10-7.32(m, 5H).

EXAMPLE 9

Preparation of (1,1'-biphenyl-4-yl)-L-alanine methyl ester hydrochloride

Thionyl chloride (0.043 ml; 0.586 mmoles) was added at room temperatureto a solution of N-tert-butoxycarbonyl-(1,1'-biphenyl-4-yl)-L-alanine(100 mg; 0.293 mmoles) in methanol (2 ml).

After 24 hours, the reaction mixture was concentrated at small volume byevaporation under vacuum affording (1,1'-biphenyl-4-yl)-L-alanine methylester hydrochloride with a practically quantitative yield (85 mg) as acrystalline solid.

m.p. 215°-216° C.

¹ H-NMR (200 MHz, DMSO-d₆): δ (ppm): 3.15 (dd, 2H); 3.70 (s, 3H); 4.30(t, 1H); 7.25-7.52 (m, 5H); 7.65 (m, 4H).

EXAMPLE 10

Preparation of N- 3-(3-phenylpropyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester

A solution of dicyclohexylcarbodiimide (0.433 g: 2.1 mmoles) in dioxane(15 ml) was added, at 0° C. and under stirring, to a solution of 3-(3-phenylpropyl)(methoxy)phosphinyl!-2-isobutyl-propionic acid (0.62 g;2.1 mmoles), prepared as described in example 8, andN-hydroxysuccinimide (0.242 g; 2.1 mmoles) in dioxane (15 ml).

After two hours at room temperature, dicyclohexylurea was filtered offand (1,1'-biphenyl-4-yl)-L-alanine methyl ester hydrochloride (0.56 g;1.91 mmoles), prepared as described in example 9, and triethylamine(0.266 ml; 1.91 mmoles) were added to the resultant solution.

After keeping fop one night at room temperature and heating at 60° C.for 4 hours, the reaction mixture was diluted with water and extractedwith ethyl acetate.

After drying on sodium sulphate, the resultant crude from theevaporation of the solvent was silica gel column chromatographed (eluentCH₂ Cl₂ :CH₃ OH═95:5) affording N- 3-(3-phenylpropyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester (0.24 g; 22.3% yield), which was used as such in thesubsequent reaction without further purifications.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.76 (d, 6H); 1.00-1.40 (m, 3H);1.47-1.74 (m, 3H); 1.75-1.98 (m, 2H); 2.00-2.28 (m, 1H); 2.53 (m, 1H);2.64 (t, 2H); 3.03 (dd, 1H); 3.18 (dd, 1H); 3.57 (d, 3H); 3.72 (s, 3H);4.87 (q, 1H); 7.10-7.60 (m, 14H).

By working in a similar way the following compounds were prepared:

N- 3-(4-phenylbutyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.70-0.90 (m, 6H); 1.00-1.80 (m,10H); 1.85-2.20 (m, 1H); 2.50-2.70 (m, 3H); 3.00-3.25 (m, 2H); 3.46-3.65(m, 3H); 3.68 (m, 3H); 4.87 (m, 1H); 7.00-7.60 (m, 14H).

N-(4-phenylbutyl)(methoxy)phosphinyl!acetyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 1.50-1.90 (m, 6H); 2.55 (m, 2H); 2.77(m, 2H); 3.04 (dd, 1H); 3.22 (m, 1H); 3.54, 3.67 (dd, 3H); 3.72 (s, 3H);4.84 (m, 1H); 7.00-7.60 (m, 14H).

By working in a similar way and by using 3-(benzyloxycarbonylaminomethyl) (methoxy)phosphinyl!-2-isobutyl-propionicacid as starting material, prepared as described in J. Am. Chem. Soc.1991, 113, 297-307, the following compound was prepared:

N- 3-(benzyloxycarbonylaminomethyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.80-0.90 (m, 6H); 1.00-2.30 (m, 5H);2.50-2.80 (m, 1H); 3.12 (bd, 2H); 3.30-3.80 (m, 8H); 4.86 (m, 1H); 5.07(dd, 3H); 7.18-7.58 (m, 14H).

EXAMPLE 11

Preparation of N-(2-isobutyl-acryloyl)-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester

A suspension of (1,1'-biphenyl-4-yl)-L-alanine methyl esterhydrochloride (7.03 g; 24 mmoles), prepared as described in example 9,2-isobutyl-acrylic acid (3.7 g; 28.9 mmoles), triethylamine (4.01 ml;28.9 mmoles) and dicyclohexylcarbodiimide (5.95 g; 28.9 mmoles) inmethylene chloride (140 ml) was kept under stirring at room temperaturefor 21 hours.

The resultant precipitate was filtered off and the organic phase waswashed with water, dried on sodium sulphate and evaporated at reducedpressure.

The resultant crude was purified by silica gel column chromatography(eluent 40°-60° C. petroleum ether: ethyl acetate=80:20) affordingN-(2-isobutyl-acryloyl)-(1,1'-biphenyl-4-yl)-L-alanine methyl ester(4.17 g; 47% yield) as a white solid.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.93 (d, 6H); 1.50-1.75 (m, 1H); 2.15(d, 2H); 3.08-3.30 (m, 2H); 3.75 (s, 3H); 4.95 (m, 1H); 5.21 (s, 1H);5.58 (s, 1H); 7.00-7.60 (m, 9H).

By working in a similar way the following compound was prepared:

N-(2-isopropyl-acryloyl)-(1,1'-biphenyl-4-yl)-L-alanine methyl ester

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 1.00 (m, 6H); 2.68-2.85 (m, 1H);3.05-3.31 (m, 2H); 3.75 (s, 3H); 4.90-5.02 (m, 1H); 5.20 (s, 1H); 5.41(s, 1H); 6.18 (d, 1H); 7.15-7.65 (m, 9H).

EXAMPLE 12

Preparation of 1,3,5-tris 2-phenylethyl!-hexahydrotriazine

2-Phenylethylamine (57.7 ml) was added to an aqueous solution offormaldehyde at 40% (34.3 ml; 0.457 moles), keeping the temperaturebelow 20° C.

The reaction mixture was kept under stirring at room temperature for 24hours.

After that, methylene chloride was therein added and the reactionmixture was washed with water.

The phases were separated and the organic phase was dried on sodiumsulphate and evaporated at reduced pressure obtaining 1,3,5-tris2-phenylethyl!-hexahydrotriazine (57 g; 94% yield) as an oil which wasused as such in the subsequent reaction without further purifications.

EXAMPLE 13

Preparation N-(diethoxy-methyl)(ethoxy)phosphinyl!methyl!-2-phenylethylamine

A mixture of 1,3,5-tris 2-phenylethyl!-hexahydrotriazine (3.4 g; 8.5mmoles), prepared as described in example 12, and ethyl(diethoxy-methyl)phosphinic acid (3.4 g; 25.5 mmoles), prepared asdescribed in Aust. J. Chem., 1980, 33, 287-94, in toluene (50 ml) washeated at reflux for 2 hours.

The reaction mixture was evaporated under vacuum obtaining N-(diethoxy-methyl)(ethoxy)phosphinyl!methyl!-2-phenylethylamine (8.4 g)as a colorless oil which was used as such in the subsequent reactionwithout further purifications.

EXAMPLE 14

Preparation of (2-phenylethylaminomethyl)phosphinic acid

A mixture of N-(diethoxy-methyl)(ethoxy)phosphinyl!methyl!-2-phenylethylamine (24.9mmoles), prepared as described in example 13, in concentratedhydrochloric acid (40 ml) was heated at reflux for 2 hours.

The reaction mixture was evaporated at reduced pressure and the residuewas collected with ethyl acetate and filtered.

The thus obtained solid was dissolved in warm methanol (50 ml) andfiltered.

The resultant solution was then treated with an excess of propyleneoxide and after 45 minutes a precipitate was formed.

The precipitate was filtered and dried under vacuum at 40° C. affording(2-phenylethylaminomethyl)phosphinic acid (2.07 g; 42% yield) as a whitesolid which was used as such in the subsequent reaction without furtherpurifications.

¹ H-NMR (200 MHz, D₂ O): δ (ppm): 2.85 (t, 2H); 2.95 (dd, 2H); 3.25 (t,2H); 5.60 (t, 0.5H); 7.10-7.30 (m, 5H); 8.33 (t, 0.5H).

EXAMPLE 15

Preparation of (N-benzyloxycarbonyl-2-phenylethylaminomethyl)phosphinicacid

Benzylchloroformate (1.94 ml; 12.35 mmoles) and an aqueous solution ofsodium hydroxide 1N (12.35 ml; 12.35 mmoles) were simultaneously added,below 5° C. and under nitrogen atmosphere, to a solution of(2-phenylethylaminomethyl)phosphinic acid (2.05 g; 10.29 mmoles),prepared as described in example 14, in an aqueous solution of sodiumhydroxide 1N (10.29 ml; 10.29 mmoles).

The reaction mixture was kept under stirring at room temperature for onehour.

After that, water (20 ml) was therein added and the mixture was washedwith ethyl acetate and acidified with hydrochloric acid. The reactionmixture was extracted with ethyl acetate and the organic phase waswashed with water and dried on sodium sulphate. By evaporating thesolvent at reduced pressure,(N-benzyloxycarbonyl-2-phenylethylaminomethyl)phosphinic acid (3.3 g;46% yield) was obtained as a viscous oil which was used as such in thesubsequent reaction without further purifications.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 2.80 (m, 2H); 3.33 (t, 2H); 3.57 (m,2H); 5.10 (d, 2H); 5.61 (d, 0.5H); 7.00-7.40 (m, 10H); 8.50 (d, 0.5H).

EXAMPLE 16

Preparation of methyl(N-benzyloxycarbonyl-2-phenylethylaminomethyl)phosphinic acid

A slight excess of an etheral solution of diazomethane was added at 0°C. to a solution of(N-benzyloxycarbonyl-2-phenylethylaminomethyl)-phosphinic acid (3.3 g;9.9 mmoles), prepared as described in example 15, in methylene chloride(30 ml) up to the formation of a persistent yellow solution.

Acetic acid was therein added up to decoloration of the reaction mixturewhich was then washed with water.

The collected organic phase was dried on sodium sulphate and evaporatedat reduced pressure affording methyl(N-benzyloxycarbonyl-2-phenylethylaminomethyl)phosphinic acid (3.25 g;94% yield) as a clear oil.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 2.82 (m, 2H); 3.30-3.80 (m, 7H); 5.12(d, 2H); 5.65 (d, 0.5H); 7.00-7.40 (m, 10H); 8.46 (d, 0.5H).

EXAMPLE 17

Preparation of N- 3-(N'-benzyloxycarbonyl-2-phenylethylaminomethyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester

A solution of sodium hydroxide at 55% (0.395 g; 9.07 mmoles) was addedto a solution of methyl(N-benzyloxycarbonyl-2-phenylethyl-aminomethyl)phosphinic acid (3.15 g;9.07 mmoles), prepared as described in example 16, andN-(2-isobutyl-acryloyl)-(1,1'-biphenyl-4-yl)-L-alanine methyl ester(3.31 g; 9.07 mmoles), prepared as described in example 11, indimethylformamide (50 ml) at 5° C. The reaction mixture was kept understirring at room temperature for 6 hours and then was poured in watercontaining concentrated hydrochloric acid (2 ml).

By extracting with ethyl acetate, washing the organic phase with water,drying on sodium sulphate and evaporating at reduced pressure, a crudeoil was obtained which was purified by silica gel column chromatography(eluent ethyl acetate) affording N- 3-(N'-benzyloxycarbonyl-2-phenylethylaminomethyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester (1.66 g; 26% yield) as a colorless oil.

¹ H-NMR (200 MHz, CDCl₃ +D₂ O): δ (ppm): 0.72 (d, 6H); 1.00-1.35 (m,2H); 1.45-1.90 (m, 2H); 1.95-2.20 (m, 1H); 2.40-3.80 (m, 15H); 4.80-5.15(m, 3H); 7.00-7.60 (m, 19H).

By working in a similar way the following compound was prepared:

N- 3-(4-phenylbutyl)(methoxy)phosphinyl!-2-isopropyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.70-0.90 (m, 6H); 1.50-2.40 (m, 9H);2.50-2.65 (m, 3H); 3.00-3.25 (m, 2H); 3.50-3.70 (m, 6H); 4.80-5.00 (m,1H); 6.45-6.55 (m, 1H); 7.03-7.60 (m, 14H).

EXAMPLE 18

Preparation of N- 3-(3-phenylpropyl)(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninedilithium salt (Compound 1)

A solution of N- 3-(3-phenylpropyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester (0.24 g; 0.43 mmoles), prepared as described in example 10,and lithium hydroxide monohydrate (54 mg; 1.27 mmoles) in awater:tetrahydrofuran=1:2 mixture was kept at room temperature for 72hours.

After evaporation of the solvent under vacuum to small volume, theresidue was collected with water (5 ml) and the aqueous solution waswashed with ethyl acetate (2×10 ml).

The aqueous phase was acidified with hydrochloric acid 1N.

After extracting with ethyl acetate and drying on sodium sulphate, thesolvent was evaporated.

The crude was silica Eel column chromatographed (eluent CH₂ Cl₂ :CH₃OH:CH₃ COOH═90:10:1) affording N- 3-(3-phenylpropyl)(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl-L-alaninedilithium salt (70 mg; 31% yield) as an oil which tends to solidify.

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.63 (t, 3H); 0.77 (t, 3H); 0.80-2.20(m, 11H); 2.57 (g, 2H); 2.71 (m, 1H); 2.86-3.45 (m, 2H); 4.76 (m, 0.5H);4.98 (m, 0.5H); 7.00-7.60 (m, 14H).

By working in a similar way the following compounds were prepared:

N- 3-(4-phenylbutyl)(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninedisodium salt (Compound 2)

¹ H-NMR (200 MHz, D₂ O): δ (ppm): 0.29-0.69 (m, 6H); 0.78-1.63 (m, 11H);2.17-2.51 (m, 3H); 2.63-3.19 (m, 2H); 4.30-4.48 (m, 1H); 6.90-7.45 (m,14H).

N-(4-phenylbutyl)(hydroxy)phosphinyl!-acetyl!-(1,1'-biphenyl-4-yl)-L-alaninedisodium salt (Compound 3)

¹ H-NMR (200 MHz, D₂ O): δ (ppm): 0.91-1.25 (m, 6H); 2.20 (m, 2H);2.24-2.58 (m, 2H); 2.77 (dd, 1H); 3.13 (dd, 1H); 4.33 (dd, 1H);6.80-7.50 (m, 14H).

N- 3-(4-phenylbutyl)(hydroxy)phosphinyl!-2-isopropyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninedisodium salt (Compound 4)

¹ H-NMR (200 MHz, D₂ O): δ (ppm): 0.38-0.76 (m, 6H); 0.82-2.22 (m, 9H);2.35-2.42 (m, 2H); 2.34-2.49 (m, 1H); 2.69-3.16 (m, 2H); 4.19-4.26,4.40-4.47 (2m, 1H); 6.89-7.51 (m, 14H).

EXAMPLE 19

Preparation of N- 3-(N'-benzyloxycarbonyl-2-phenylethylaminomethyl)(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alanine

A solution of N- 3-(N'-benzyloxycarbonyl-2-phenylethylaminomethyl)-(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester (1.64 g; 2.3 mmoles), prepared as described in example 17,and lithium hydroxide monohydrate (0.386 g; 9.2 mmoles) in awater:tetrahydrofuran=1:1 mixture (60 ml) was kept under stirring atroom temperature for 24 hours and under nitrogen atmosphere. Thereaction mixture was diluted with water and treated with concentratedhydrochloric acid (1 ml).

The mixture was then extracted with ethyl acetate and the organic phasewas dried on sodium sulphate and evaporated under vacuum affording N- 3-(N'-benzyloxycarbonyl-2-phenylethylaminomethyl)(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alanine(1.55 g; 98% yield).

¹ H-NMR (200 MHz, DMSO-d₆): δ (ppm): 0.60 (d, 3H); 0.68 (d, 3H);0.90-1.95 (m, 5H); 2.64 (m, 1H); 2.83 (t, 2H); 2.90 (dd, 1H); 3.20 (dd,1H); 3.45-3.65 (m, 4H); 4.60 (m, 1H); 5.05 (s, 2H); 7.10-7.65 (m, 19H).

EXAMPLE 20

Preparation of N- 3-(2-phenylethylaminomethyl)(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alanine(Compound 5)

A solution of N- 3-(N'-benzyloxycarbonyl-2-phenylethylaminomethyl)-(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alanine(1.53 g; 2.23 mmoles), prepared as described in example 19, in ethanolat 80% (100 ml) was hydrogenated into a Parr apparatus in the presenceof palladium on charcoal at 10% (0.3 g).

When the hydrogen absorption was over, the catalyst was filtered off andby evaporating the resultant solution under vacuum a crystalline productwas formed.

After filtration and drying under vacuum at 40° C. N- 3-(2-phenylethylaminomethyl)(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alanine(0.9 g; 73% yield) was thus obtained as a white solid.

m.p. 229°-230° C.

¹ H-NMR (200 MHz, DMSO-d₆): δ (ppm): 0.63 (d, 3H); 0.72 (d, 3H); 1.05(m, 2H); 1.30-1.85 (m, 3H); 2.60 (m, 1H); 2.80-3.32 (m, 8H); 4.62 (m,1H); 7.20-7.61 (m, 14H).

EXAMPLE 21

Preparation of N- 3-(aminomethyl)(methoxy)phosphinyl!-2-isobutylpropionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester hydrochloride

A solution of N- 3-(benzyloxycarbonylaminomethyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester (1.75 g; 2.87 mmoles), prepared as described in example 10,and hydrochloride acid 12N (0.24 ml; 2.87 mmoles) in methanol (50 ml)was hydrogenated into a Part apparatus at room temperature in thepresence of catalytic amounts of palladium on charcoal.

The catalyst was then filtered off and the solvent was evaporated atreduced pressure affording N- 3-(aminomethyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester hydrochloride (1.43 g, 97% yield).

¹ H-NMR (200 MHz, D₂ O): δ (ppm): 0.56-0.70 (2d, 6H); 1.00-1.40 (m, 3H);1.80-2.03 (m, 2H); 2.45-2.70 (m, 1H); 2.80-3.20 (m, 4H); 3.35-3.52 (2d,3H); 3.55 (s, 3H); 4.55 (m, 1H); 7.12-7.50 (m, 9H).

EXAMPLE 22

Preparation of N- 3-(benzoylaminomethyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester

Benzoyl chloride (0.191 ml; 1.64 mmoles) and triethylamine (0.457 ml;3.29 mmoles) were added at room temperature and under stirring to asolution of N- 3-(aminomethyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester hydrochloride (0.7 g; 1.37 mmoles), prepared as describedin example 21, in methylene chloride (20 ml).

After 30 minutes the reaction mixture was washed with an aqueoussolution of potassium bisulphite at 2% (10 ml), water, an aqueoussolution of sodium bicarbonate at 2% (10 ml) and water, respectively.

The collected organic phase was dried on sodium sulphate and evaporatedat reduced pressure.

The residue was purified by silica gel column chromatography (eluentmethylene chloride:methanol=97:3) affording N- 3-(benzoylaminomethyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alaninemethyl ester (0.66 g; 83% yield).

¹ H-NMR (200 MHz, CDCl₃): δ (ppm): 0.78-0.94 (2d, 6H); 1.20-2.35 (m,5H); 2.60-2.90 (m, 1H); 2.95-3.26 (m, 2H); 3.60-4.30 (m, 8H); 4.75-5.00(m, 1H); 7.12-7.55 (m, 12H); 7.83 (d, 2H).

EXAMPLE 23

Preparation of N- 3-(benzoylaminomethyl)(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alanine(Compound 6)

By working as described in example 19 and by using N- 3-(benzoylaminomethyl)(methoxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alanine methyl ester (0.64 g;1.11 mmoles), prepared as described in example 22, N- 3-(benzoylaminomethyl)(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alanine(0.56 g; 92% yield) was obtained as a white crystalline solid.

m.p. 148°-150° C.

¹ H-NMR (200 MHz, D₂ O+NaHCO₃): δ (ppm): 0.60 (d, 3H); 0.66 (d, 3H);1.08-1.60 (m, 5H) 2.41-2.60 (m, 1H); 2.60-3.25 (m, 4H); 4.35-4.42 (m,1H); 7.08-7.50 (m, 14H).

By working in a similar way the following compound was prepared:

N- 3-(phenylacetylaminomethyl)(hydroxy)phosphinyl!-2-isobutyl-propionyl!-(1,1'-biphenyl-4-yl)-L-alanine(Compound 7)

m.p. 176°-177° C.

¹ H-NMR (200 MHz, D₂ O+NaHCO₃): δ (ppm): 0.59, 0.65 (2d, 6H); 1.08-1.45(m, 5H); 2.32-2.47 (m, 1H); 2.50-3.11 (m, 4H); 3.26 (s, 2H); 4.32-4.39(m, 1H); 7.04-7.46 (m, 14H).

EXAMPLE 24

"In vitro" evaluation of the pharmacologic activity

a) NEP-inhibitory activity

The NEP-inhibitory activity was evaluated according to the methodreported in the literature by C. Llorens et al., in Eur. J. Pharmacol.,69, (1981), 113-116.

Membranes from kidney cortex were prepared according to the followingprocedure.

By working at 0°-4° C., kidneys were removed from male Sprague-Dawleyrats weighing approximately 300 g.

Cortex was carefully dissected, finely minced and suspended in ahomogenization buffer (10 mM sodium phosphate pH 7.4 containing 1 mMMgCl₂, 30 mM NaCl, 0.02% NAN₃) 1:15 weight/volume.

The tissue was then homogenized for 30 seconds using an Ultra-Turraxhomogenizer.

Approximately 10 ml of homogenate were layered over 10 ml of sucrose(41% weight/volume) and centrifuged at 31200 rpm for 30 minutes at 4° C.in a fixed angle rotor.

The membranes were collected from the buffer/sucrose interface, washedtwice with 50 mM TRIS/HCl buffer (pH 7.4) and resuspended into the samebuffer for storage.

The membranes were stored in small aliquots at -80° C. until use. TheNEP-inhibitory activity was evaluated by using the following method.

Aliquots of the membrane suspension prepared as above described(concentration 5 μg/ml of proteins) were preincubated in the presence ofan aminopeptidase inhibitor (Bestatin--1 mM) for 10 minutes at 30° C.

³ H! Leu⁵ !-enkephaline (15 nM) and buffer TRIS/HCl pH 7.4 (50 mM) wereadded in order to obtain a final volume of 100 μl. Incubation (20minutes at 30° C.) was stopped by adding HCl 0.1M (100 μl).

The formation of the metabolite ³ H!Tyr-Gly-Gly was quantified bychromatography on polystyrene columns (Porapak Q). The percentage ofinhibition of the metabolite formation in the membrane preparationstreated with the compounds of formula I, in comparison to the untreatedmembrane preparations, was expressed as IC₅₀ value (nM).

b) ACE-inhibitory activity

The ACE-inhibitory activity was evaluated according to the methodreported in the literature by B. Holmquist et al., in AnalyticalBiochemistry 95, 540-548 (1979).

50 μM of ACE (250 mU/ml purified by lung rabbit, EC 3.4.15.1 SIGMA) with50 μl of the compound of formula I were preincubated in thermostatedcuvettes at 37° C.

The reaction was started by addingfurylacryloylphenylalanylglycylglycine 0.8 mM (FAPGG-SIGMA).

Contemporaneously, by using a Beckman DU-50 spectrophotometer providedwith a program for calculating delta A/minutes and regressioncoefficients of the enzyme kinetics curves, the absorbance at 340 nm wasrecorded in continuo for 5 minutes.

The percentage of the enzyme inhibition in the preparations treated withthe compounds of formula I with respect to the untreated preparationswas expressed as IC₅₀ value (nM).

The compounds of formula I were tested in the form of lithium or sodiumsalts.

We report in the following table 1 the IC₅₀ values (nM) related to theACE-inhibitory activity and NEP-inhibitory activity of compounds 1, 2, 6and 7.

                  TABLE 1                                                         ______________________________________                                        ACE-inhibitory and NEP-inhibitory activity of compound 1, compound            2, compound 6 and compound 7 expressed as IC.sub.50 (nM).                              ACE-inhibitory activity                                                                       NEP-inhibitory activity                              Compound IC.sub.50 (nM)  IC.sub.50 (nM)                                       ______________________________________                                        1        70              368                                                  2        24              220                                                  6        4.6             7.7                                                  7        183.0           30.1                                                 ______________________________________                                    

The data reported in table 1 show that the compounds of formula I,object of the present invention, are endowed with a significant mixedACE/NEP inhibitory activity.

We claim:
 1. A compound of formula ##STR11## wherein R is a biphenylgroup optionally substituted by one or more substituents, the same ordifferent, selected among halogen atoms, hydroxy groups, alkoxy, alkyl,thioalkyl or alkoxycarbonyl groups having from 1 to 6 carbon atoms inthe alkyl moiety, C₁ -C₃ alkyl groups containing one or more fluorineatoms, carboxy groups, nitro groups, amino or aminocarbonyl groups,acylamino groups, aminosulphonyl groups, mono- or di-alkylamino groupshaving from 1 to 6 carbon atoms in the alkyl moiety, mono- ordi-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in thealkyl moiety;R₁ is a hydrogen atom, a straight or branched C₁ -C₆ alkylgroup or an arylalkyl group having from 1 to 6 carbon atoms in the alkylmoiety wherein the aryl is a phenyl, a biphenyl, a naphthyl or a 5 or 6membered aromatic heterocycle with one or two heteroatoms selected amongnitrogen, oxygen and sulphur, optionally substituted with one or moresubstituents, the same or different, selected among halogen atoms,hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups havingfrom 1 to 6 carbon atoms in the alkyl moiety, C₁ -C₃ alkyl groupscontaining one or more fluorine atoms, carboxy groups, nitro groups,amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups,mono- or di-alkylamino groups having from 1 to 6 carbon atoms in thealkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6carbon atoms in the alkyl moiety; R₂ is a straight or branched C₁ -C₆alkyl group, optionally containing one or more fluorine atoms or one ormore --NH-- groups, an arylalkyl, an arylcarbonylaminoalkyl, anarylalkylcarbonylaminoalkyl or an arylaminocarbonylalkyl group havingfrom 1 to 6 carbon atoms and optionally one or more --NH-- groups in thealkyl moiety, the aryl being optionally substituted by one or moresubstituents, the same or different, selected among halogen atoms,hydroxy groups, alkoxy, alkyl, thioalkyl or alkoxycarbonyl groups havingfrom 1 to 6 carbon atoms in the alkyl moiety, C₁ -C₃ alkyl groupscontaining one or more fluorine atoms, carboxy groups, nitro groups,amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups,mono- or di-alkylamino groups having from 1 to 6 carbon atoms in thealkyl moiety, mono- or di-alkylaminocarbonyl groups having from 1 to 6carbon atoms in the alkyl moiety; m is 0 or 1; X is a hydrogen orfluorine atom;the carbon atom marked with an asterisk is an asymmetriccarbon atom; and pharmaceutically acceptable salts thereof.
 2. Acompound according to claim 1 wherein R is a biphenyl group optionallysubstituted with from 1 to 3 substituents, the same or different,selected among chlorine or fluorine atoms or hydroxy groups; R₁ is astraight or branched C₁ -C₆ alkyl group or an arylalkyl group havingfrom 1 to 6 carbon atoms in the alkyl moiety wherein the aryl group is aphenyl or a bipheny; R₂ is a straight or branched C₁ -C₆ alkyl, anarylcarbonylaminoalkyl, an arylalkylcarbonylaminoalkyl or an arylalkylgroup having from 1 to 6 carbon atoms in the alkyl moiety wherein thearyl group is a phenyl, m is 1 and X is hydrogen.
 3. A compoundaccording to claim 2 wherein R is a 4-biphenyl group; R₁ is a straightor branched C₃ -C₆ alkyl group; R₂ is an arylcarbonylaminoalkyl or anarylalkylcarbonylaminoalkyl group having from 1 to 3 carbon atoms in thealkyl moiety wherein the aryl group is a phenyl, m=1 and X=H.
 4. Acompound according to claim 1 in the form of a salt with an alkali metalselected among sodium, lithium and potassium.
 5. A process for thepreparation of a compound of formula I according to claim 1 whichcomprises the reaction between a compound of formula ##STR12## whereinR₁, R₂, m and X have the meanings reported in claim 1 and Y represents aprotective group selected among a C₁ -C₄ alkyl, a phenyl or aphenylalkyl group having from 1 to 4 carbon atoms in the alkylmoiety;and a biphenylalanine derivative of formula ##STR13## wherein Rhas the meanings reported in claim
 1. 6. A process for the preparationof a compound of formula I wherein m=1 and X=H according to claim Iwhich comprises the reaction between a compound of formula ##STR14##wherein R₂ has the meanings reported in claim 1 and Y represents aprotective group selected among a C₁ -C₄ alkyl, a phenyl or aphenylalkyl group having from 1 to 4 carbon atoms in the alkyl moiety;and a compound of formula ##STR15## wherein R and R₁ have the meaningsreported in claim
 1. 7. A pharmaceutical composition containing atherapeutically effective amount of a compound of formula I according toclaim 1 in admixture with a carrier for pharmaceutical use.
 8. Apharmaceutical composition according to claim 7 for the treatment ofcardiovascular diseases.